Screening for Antimicrobial Resistance and Genes of Exotoxins in Pseudomonas aeruginosa Isolates from Infected Dogs and Cats in Poland

Pseudomonas aeruginosa has assumed an increasingly prominent role as the aetiological agent in serious hard-to-treat infections in animals and humans. In this study, 271 P. aeruginosa strains collected from dogs and cats were investigated. The aim of the research was to screen these P. aeruginosa strains for antibiotic resistance and the presence of selected virulence factor genes. Antibiotic resistance was determined using the Kirby–Bauer method, while virulence genes were detected by polymerase chain reaction (PCR). The most frequently detected resistance was to fluoroquinolones, ranging in prevalence from 17.3% for ciprofloxacin up to 83% for enrofloxacin. The resistance to carbapenems was 14% and 4.8% for imipenem and meropenem, respectively. Almost all P. aeruginosa strains harboured the exoT (97.8%) and lasB (93.4%) genes, while the lowest prevalence was found for exoU (17.3%) and plcH (17.3%). P. aeruginosa strains isolated from dogs that harboured the toxA gene were more frequently resistant to ceftazidime (p = 0.012), while the presence of the exoU gene was found to be connected with resistance to marbofloxacin (p = 0.025) and amikacin (p = 0.056). In strains originating from cats, only the connection between the presence of the exoU gene and resistance to enrofloxacin (p = 0.054) was observed. The confirmation of associations between virulence-factor-encoding genes and antibiotic resistance indicates that problems of antibiotic resistance may not only cause complications at the level of antibiotic dosage but also lead to changes in the virulence of the bacteria; thus, further studies in this area are required.


Introduction
Pseudomonas aeruginosa (P. aeruginosa) is an oxidase-positive Gram-negative bacillus with minimal nutrient requirements, a feature that, combined with the possibility of its growth across a broad range of temperature (4-44 • C), is recognized to contribute to its ubiquity [1]. In nature, P. aeruginosa can exist in biofilm, attached to a surface or substrate, or in planktonic form, in which it can actively move via its flagellum [2]. P. aeruginosa is an opportunistic bacterium and very rarely causes infection in healthy individuals; however, in certain circumstances, it can directly threaten the health or the life of an individual [1,3,4]. Nevertheless, due to its high resistance level to antimicrobials and disinfectants, P. aeruginosa is a major cause of nosocomial infections in humans (7.1% in European countries) [5,6]. The risk factors to humans associated with P. aeruginosa infections are chronic obstructive pulmonary disease, diabetes, cystic fibrosis, immunosuppression (after organ or bone marrow transplantation), severe kidney and liver failure, and multiorgan failure [7][8][9]. In dogs the most frequent P. aeruginosa is isolated from patients with ear infections, nevertheless, the bacteria can cause a range of other infections including skin infections, urinary tract infections, and from phosphatidylserine and phosphatidylcholine; and plcH (haemolytic phospholipase C) releases phosphate esters from sphingomyelin and phosphatidylcholine [26].
The current study aims to screen and compare the distribution of selected virulence factor genes and antibiotic resistance in P. aeruginosa strains of pet origin. The results of this study will help guide empirical antimicrobial selection for the treatment of dogs and cats infected with P. aeruginosa in veterinary medicine. In addition, the data generated from this study will contribute to antimicrobial resistance surveillance programmes in animal health.

Isolates
This study includes 271 P. aeruginosa isolates collected from cats (n = 59) and dogs (n = 212) from the Lower Silesia area between 2017 and 2020 in the commercial veterinary laboratory, VETLAB Ltd., in Wrocław, Poland. The bacterial isolates were transferred to the Department of Epizootiology and Clinic of Birds and Exotic Animals, Wrocław University of Environmental and Life Sciences. The isolates were cultured on the Columbia Blood Agar Base (OXOID, Basingstoke, UK) with 5% sheep blood and MacConkey Agar (OXOID, Basingstoke, UK). Then isolates were checked for oxidase production (Oxidase Detection Strips MICROGEN MID-61g GRASO BIOTECH, Starogard Gdański, Poland). Pure strains of nonfermenting bacilli, suspected to be P. areuginosa strains were frozen in 1 mL of Brain Heart Infusion Broth (BHI) (OXOID, Basingstoke, UK) supplemented with 30% glycerol and storage in −80 °C for up to six months for further analysis. Subsequently they were used for molecular investigation. Belonging to the P. aeruginosa species was provided by polymerase chain reaction (PCR). The details of procedure used for species identification of the P. aeruginosa strains used in the current study is described in Płókarz et al. (2022) [27]. The bacterial strains originated from pets with clinical manifestations of respiratory tract infections (swabs from the nasal cavity, conjunctival sacs, oral cavity, throat, trachea, and bronchi), skin, wounds, joint fluid, and genitourinary tract. Details are included in Figures 1 and 2.

Detection of Virulence Genes
The presence of toxin genes and occurrence of other virulence factors genes was assayed in DNA isolated from the P. aeruginosa strains. Isolation of DNA was carried out as previously described by Płókarz et al. (2022) [27]. The DNA was amplified in a thermocycler (Bio-Rad, Marnes-la-Coquette, France) in 2.5 µL buffer (MgCl2 at 20 mM concentration), 0.2 µL Taq DNA polymerase (5 U/L) (Thermo Fisher Scientific, Vilnius, Lithuania), 0.2 µL dNTP mix 10 mM concentration (Thermo Fisher Scientific, Vilnius, Lithuania), 0.2 µL of each specific primer, and 2 µL template DNA. The reaction volume was adjusted to 25 µL with sterile water. The used primers and cycle conditions are presented in Table 1.

Detection of Virulence Genes
The presence of toxin genes and occurrence of other virulence factors genes was assayed in DNA isolated from the P. aeruginosa strains. Isolation of DNA was carried out as previously described by Płókarz et al. (2022) [27]. The DNA was amplified in a thermocycler (Bio-Rad, Marnes-la-Coquette, France) in 2.5 µL buffer (MgCl 2 at 20 mM concentration), 0.2 µL Taq DNA polymerase (5 U/L) (Thermo Fisher Scientific, Vilnius, Lithuania), 0.2 µL dNTP mix 10 mM concentration (Thermo Fisher Scientific, Vilnius, Lithuania), 0.2 µL of each specific primer, and 2 µL template DNA. The reaction volume was adjusted to 25 µL with sterile water. The used primers and cycle conditions are presented in Table 1.

Statistical Analysis
Statistical analysis was performed using TIBCO Statistica 13.3 (TIBCO Software Inc., Palo Alto, CA, USA). Spearman's rank correlation coefficient was used to analyse the correlation between antibiotic resistance and the origin P. aeruginosa isolates Wilson's test was used to establish the confidence interval (95% PU). The chi-square test and Fisher's exact test were used to determine the significant difference in the virulence factor's gene occurrence between P. aeruginosa strains susceptible and resistant to selected antibiotics. The statistical significance level was configured as p < 0.05.

Antibiotic Resistance
In the current study, the resistance of 271 P. aeruginosa strains to specific antibiotics was investigated. In the beginning, the differences between the antibiotic resistance of P. aeruginosa originating from dogs or cats were tested. Only in the fluoroquinolone class significant differences between dogs and cats occur. In dogs, P. aeruginosa isolates were statistically significantly more often resistant to fluoroquinolone than isolates from cats (p ≤ 0.001). In other groups, there were no statistically significant differences. In P. aeruginosa strains from both dogs and cats, the most frequently detected resistance was to fluoroquinolones (from 17.3% (CI 95%: 13.3-22.3%) for ciprofloxacin to 83% (CI 95%: 78.1-87%) for enrofloxacin). Among the aminoglycosides, the highest resistance was observed to gentamicin at 26.9% (CI 95%: 22-32.5%), whereas in the carbapenem class, resistance to imipenem (14% (CI 95%: 8.2-15.8%)) was around two times higher than to meropenem (4.8% (CI 95%: 2.8-8%)). The detailed data are presented in Figure 3.    In the P. aeruginosa strains from dogs, there were some statistically significant correlations between the resistance to a particular antibiotic and the material from which bacteria was isolated; for example, the strains from wounds were frequently resistant to ticarcillin (p = 0.014), the strains from the urogenital system to meropenem (p = 0.042), the strains from the external auricular canals to marbofloxacin (p = 0.016), and from the skin and appendages to ciprofloxacin (p = 0.023) and gentamicin (p = 0.022). The detailed data  Table 2. In cats, this correlation occurred only in the case of tobramycin, where strains from the respiratory system were significantly less resistant to this antibiotic (p = 0.026) ( Table 3).

Virulence Factor Genes
Almost all P. aeruginosa strains harboured the exoT (97.8%) and lasB (93.4%) genes, while the lowest prevalence was found for the exoU (17.3%) and plcH (17.3%) genes. There was no observation of differences in the presence of exoenzyme genes in strains of dog or cat origin except for the occurrence of exoS, which was significantly more frequently detected in the P. aeruginosa from cats (p = 0.029). The detailed data are presented in Figure 4. There were only a few statistically significant associations between harbouring the virulence gene and exhibiting resistance to a particular antibiotic. In the P. aeruginosa strains from dogs, there were connections such as toxA and ceftazidime (p = 0.012) and exoU and marbofloxacin (p = 0.025) and amikacin (p = 0.056), while in the strains from cats, exoU and enrofloxacin (p = 0.054) were associated. The detailed data are presented in the Supplementary Materials Tables S1 and S2.
Almost all P. aeruginosa strains harboured the exoT (97.8%) and lasB (93.4%) genes, while the lowest prevalence was found for the exoU (17.3%) and plcH (17.3%) genes. There was no observation of differences in the presence of exoenzyme genes in strains of dog or cat origin except for the occurrence of exoS, which was significantly more frequently detected in the P. aeruginosa from cats (p = 0.029). The detailed data are presented in Figure  4. There were only a few statistically significant associations between harbouring the virulence gene and exhibiting resistance to a particular antibiotic. In the P. aeruginosa strains from dogs, there were connections such as toxA and ceftazidime (p = 0.012) and exoU and marbofloxacin (p = 0.025) and amikacin (p = 0.056), while in the strains from cats, exoU and enrofloxacin (p = 0.054) were associated. The detailed data are presented in the Supplementary Materials Tables S1 and S2. .

Discussion
The disc diffusion method is still a widespread way to diagnose antibiotic resistance in commercial laboratories in Poland, because it is a relatively cheap and reliable method. Previous reports have indicated good effectiveness in the confirmation of resistance to antibiotics compared with determination of the minimal inhibitory concentration method (MIC) [31]. The disc diffusion method can be useful for screening antibiotic resistance. As our research considered a large number of P. aeruginosa strains under investigation, we decided to assess antibiotic resistance using this method.
In the current study, P. aeruginosa strains were most frequently resistant to fluoroquinolones. The majority of strains, 225 from among 271, presented resistance to enrofloxacin (83%), while the P. aeruginosa strains were the most susceptible to ciprofloxacin (only

Discussion
The disc diffusion method is still a widespread way to diagnose antibiotic resistance in commercial laboratories in Poland, because it is a relatively cheap and reliable method. Previous reports have indicated good effectiveness in the confirmation of resistance to antibiotics compared with determination of the minimal inhibitory concentration method (MIC) [31]. The disc diffusion method can be useful for screening antibiotic resistance. As our research considered a large number of P. aeruginosa strains under investigation, we decided to assess antibiotic resistance using this method.
In the current study, P. aeruginosa strains were most frequently resistant to fluoroquinolones. The majority of strains, 225 from among 271, presented resistance to enrofloxacin (83%), while the P. aeruginosa strains were the most susceptible to ciprofloxacin (only 47 from among 271 (17.3%) of P. aeruginosa were resistant). In bacteria originating from cats, the resistance to ciprofloxacin was low, at 3 from among 59 P. aeruginosa strains (5.1%), compared with the P. aeruginosa strains from dogs, where 44 from among 212 were resistant (20.8%). The obtained results are similar to those previously reported by Penna et al. (2011) [32], where 13.8% of strains of P. aeruginosa were found to be resistant to ciprofloxacin, while 63.6% were resistant to enrofloxacin. In the study by Eliasy et al. (2020), where the antimicrobial resistance patterns of P. aeruginosa from clinical samples from dogs were investigated, a high proportion of strains resistant to fluoroquinolones (enrofloxacin 73% and orbifloxacin 90%) were also detected [33]. Similar tendencies were also observed in strains of human origin where the resistance to ciprofloxacin was 31.36% and the resistance to levofloxacin was 79.57% [34]. However, we observed significantly lower resistance of P. aeruginosa strains to aminoglycosides compared with the previous study by Penna et al. (2011), where the resistance of strains was 71.4% for gentamicin and 64.4% for tobramycin [32]. Accordance with our results resistance to aminoglycosides was as follows: amikacin (16%), gentamicin (18%), and tobramycin (12%). The differences in the obtained results could be caused by the origin of the bacteria. In the earlier study, only P. aeruginosa strains from the causes of canine otitis externa were investigated [32], whereas, in the current study, we analysed the material from different pathological changes in pets. Considering also that aminoglycosides are frequently used in topical therapy for external ear canal infections, the increased resistance to these antibiotics is very probable. In contrast, very low levels of P. aeruginosa strains resistant to aminoglycosides were detected in Japan, where only 4.5% of strains exhibited resistance to gentamicin and 2.5% to amikacin [35].
There were also differences in resistance levels to ceftazidime, where 58% was reported by Penna et al. (2011) [32] and 77% by Eliasi et al. (2020) [33], while in the current study 21.7% of the P. aeruginosa strains from dogs and 16.9% from cats were detected as resistant. This is in contrast to other veterinary reports, where the prevalence of resistance to ceftazidime was 0-0.5% [35][36][37]. The resistance to ticarcillin (6.6% of P. aeruginosa) in the current study is very similar to the results obtained by Pottier et al. (2022) [36] in strains from veterinary samples (7.1%). In similar studies, the reported resistance of P. aeruginosa to carbapenems was consistent with our results 6-10% [33,38,39] to imipenem and 2-6% [36,38] to meropenem. While the results provided by Dégi et al. (2021) are significantly different to the results obtained in the current study [40]. In P. aeruginosa strains isolated from dogs in Western Romania, the resistance rate was 74.14% to meropenem and 77.59% to imipenem and was significantly higher than in the current study. The reason could be that samples in the mentioned research were collected the most frequently from superficial infections while our study shows mainly samples collected from external auricular canal [40]. The resistance to carbapenems in animal P. aeruginosa strains is still at the control level and is much lower than in the bacteria that cause nosocomial infections. Occurrence of resistance against this group of antibiotics in dogs and cats are probably results of close contact between owners and pets. It suggests exchanging genetic elements such as self-transmissible conjugative plasmids through horizontal gene transfer (HGT) [41]. The recent study of the antibiotic resistance of P. aeruginosa from Polish hospitals found that 67.8% of strains are resistant to imipenem and 29.2% to meropenem [42]. According to a 2017 ECDC survey, the average resistance rate to carbapenems (imipenem + meropenem) was 24.2% across Poland [43].
Our report also describes the prevalence of the chosen virulence factor genes in the P. aeruginosa strains under investigation. Overall, the frequency determined for the exoU (17.3%) and exoS (79,7%) genes is comparable to the results obtained by Hayashi et al. (2021) of 15.8% and 82.5%, respectively [38]. Similar to our results, lasB was detected in almost all P. aeruginosa (98.7%) [44]. The high prevalence of exoT and exoY genes in the current study (over 80% of P. aeruginosa strains) was consistent with previous reports, where all or almost all strains harboured such genes (100% exoT and 89-100% exoY) [45,46].
Our results show the presence of the toxA gene in 66.4% of strains, which is significantly lower than the 91.7% obtained by Hattab et al. (2021) [47]. Significant differences were also found for the prevalence of the plcH gene, with only 17.3% found to harbour the plcH gene in our study versus 91.7% in the earlier study. This difference could be caused by the very small number of samples (n = 24) compared with our study (n = 271), which included only canine samples. In our study, 59.8% of isolates were found to possess the plcN gene, which was lower than found by Aslataş et al. (2022) in clinical cases of bovine mastitis (100%), but they examined isolates from other anatomical locations and species, and the number of samples was significantly lower [48]. It is very difficult to compare the prevalence of P. aeruginosa isolates across different studies because of the differences in examined animal species, and geographical regions, all of which can exert varying influences on the results.
The association between the presence of toxA and the antibiotic resistance of P. aeruginosa strains found in our study is supported by the earlier results of Amormozafari et al. (2016), who used the same statistical analysis [49]. The presence of exoU, exoS, exoT, and exoY was analysed in humans by Horna et al. (2019). The results of their research confirm the association between the resistance to fluoroquinolone and aminoglycoside and the occurrence of the exoU gene [45]. In our research, we found a significant association indicating that the presence of the exoU gene in P. aeruginosa strains is linked with resistance to marbofloxacin (p = 0.025) and to amikacin (p = 0.056). For the other toxin genes, however, there were no statistically significant associations. The study performed by Hayashi et al. in 2021 in canine and feline patients from Japanese animal hospitals showed a significant association of isolates possessing exoU and resistance to aminoglycoside [38].

Conclusions
Pseudomonas aeruginosa isolates tested in the current study showed the highest resistance to fluoroquinolone. This can be useful in clinical veterinary practice in experimental antibiotic therapy and suggests that veterinarians should use greater caution in the selection of antibiotics. Of note is the low resistance to aminoglycosides in cases of otitis externa, despite commercial usage of this antibiotics class as a component of ear drops. Of considerable epidemiological value, our study included an antibiogram assessment of resistance to meropenem, imipenem, and ticarcillin in isolates from dogs and cats. In our knowledge carbapenems are not used in veterinary medical practice in Poland which suggests transferring P.aeruginosa resistant strains from human to animals. Furthermore, results of research showed a higher rate of resistance in humans which supports the thesis: humans are a source of P.aeruginosa resistance strains for animals. Although the rate of the resistant strains was still low, the increasing tendency indicates control of this potential reservoir is important. The analysis of the associations between the strains possessing virulence-factor-encoding genes and antibiotic resistance allows us to broaden the horizons considering antibiotic resistance. Further research on the relationships between virulence factors and antibiotic resistance in animals is required.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antibiotics12071226/s1, Table S1: Statistical analysis of virulencefactor-encoding genes and selected antibiotics relationship in Pseudomonas aeruginosa isolates from cats; Table S2: Statistical analysis of virulence-factor-encoding genes and selected antibiotics relationship in Pseudomonas aeruginosa isolates from dogs.
Author Contributions: All authors of this research paper have directly participated in the planning and/or execution and/or analysis of this study. D.P. wrote the manuscript, provided statistical analysis, designed the research, table and figures, and analyzed data; K.B. corrected the manuscript, tables and figures; K.R. contributed to the design of the research, analyzed data and supervised the findings of this work. All authors have read and agreed to the published version of the manuscript.

Funding:
The APC/BPC is financed/co-financed by Wroclaw University of Environmental and Life Sciences.

Institutional Review Board Statement:
The samples used in this study originally came from the samples used as material for routine diagnostics.

Informed Consent Statement:
This study was provided based on material from VETLAB Ltd. in Wroclaw. All animals had their own veterinarian who was responsible for providing information about potential zoonotic risk. The study did not involve humans.

Data Availability Statement:
The datasets used and/or analyzed during the current study are available via e-mail from the corresponding author on reasonable request.